Myocilin induction of GTM cells by dexamethasone was performed as described previously.18 (link) The same publication shows myocilin induction by NTM cells used in this study. For Myo10 immunoblots, a rabbit polyclonal myosin-X primary antibody (PA5-55019; ThermoFisher, Waltham, MA, USA) and an IRDye 700-conjugated anti-rabbit secondary antibody were used. Membranes were scanned using an Odyssey gel imaging system (Licor, Lincoln, NE, USA). The intensity of the expected 260-kDa band was quantitated from each lane using ImageJ software. Also, myocilin immunostaining of GTM and NTM cells in culture was performed with and without dexamethasone treatment for 7 days. The immunostaining protocol is detailed below.
Odyssey gel imaging system
Manufactured by LI COR
Sourced in United States
The Odyssey gel imaging system is a high-performance, versatile instrument designed for the detection and quantification of fluorescent signals in a variety of samples, including protein gels, Western blots, and tissue sections. The system utilizes advanced imaging technology to provide precise and sensitive data, making it a valuable tool for researchers in various fields.
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Lab products found in correlation
Bis tris polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Irdye 700, by Thermo Fisher Scientific (1 mentions)
Pa5 55019, by Thermo Fisher Scientific (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Irdye 800 conjugated anti goat, by Rockland Immunochemicals (1 mentions)
Ab108319, by Abcam (1 mentions)
Bs6007m, by Bioworld Technology (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Rabbit beta actin, by Merck Group (1 mentions)
9 protocols using odyssey gel imaging system
1
Dexamethasone-induced Myocilin Expression
2
Cytokine and Mechanical Stretch Effects on Protein Secretion
3
Quantitative Western Blotting Assay
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Western blotting assays were performed as previously described82 (link). All antibodies used in the present study, their catalog numbers, applications, and used dilution can be found in Supplementary Table 1 . Anti-rabbit and anti-mouse IgG secondary antibodies were purchased from LI-COR. Membranes were scanned using the LI-COR Odyssey® Gel Imaging system. W.B intensities were determined using ImageJ software.
4
Protein Extraction and Immunoblotting Protocol
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Tissues were snap frozen in liquid nitrogen. Frozen tissues were lysed using RIPA buffer and phosphatase protease inhibitor (Roche, Sigma) for 30min on ice. Supernatants were stored at −80°C after samples were spun down at 12,000 rpm for 5min. Immunoblotting was as previously published5 (link). Briefly, equal amounts of protein (10μg) in 3XLaemmli buffer and 0.1M DTT were separated by electrophoresis through 4–12% Bis-Tris polyacrylamide gels (Novex, Life Technologies) and were transferred to PVDF membranes. Membranes were blocked with 1% casein block in PBS (BioRad 1610782), subsequently incubated with primary and secondary antibodies and washed with TBST (50mM Tris-HCl pH7.5, 0.15M NaCl and 0.25% Tween-20) between incubations. Blots were developed using an Odyssey Gel Imaging System (Li-Cor), and quantified using ImageStudio with Gapdh as loading control.
5
Morphine-Induced Changes in NAc Proteins
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Mice from morphine CPP/free access extinction procedure were sacrificed by decapitation at 1 h after the last extinction training and drug treatment, and the NAc tissues of five animals from each group were harvested. A Western blot assay was performed to evaluate changes in BDNF, GluA1, and GluA2 levels in the NAc of mice. Equal amounts of denatured total protein (10 µg) from each sample were separated using 12% SDS-PAGE. The separated proteins were transferred onto a 0.2 µm nitrocellulose membrane. The membrane was blocked with 5% non-fat dry milk in a TBS solution and probed with specific rabbit polyclonal GluA1 (ab31232) antibody (diluted 1:1000, Abcam), rabbit monoclonal GluA2 (ab206293) antibody (diluted 1:1000, Abcam), rabbit monoclonal BDNF (ab108319) antibody (diluted 1:1000, Abcam) overnight at 4°C. The membrane was then washed and incubated with fluorophore-conjugated donkey anti-rabbit or goat anti-mouse secondary antibody (diluted 1:10000, Rockland). The secondary antibody was light-excited (700/800 nm wavelength), and the emitted light was detected and analyzed using an Odyssey gel imaging system (LI-COR, Inc., Lincoln, NE, USA). GAPDH expression was analyzed in the same blot using a specific mouse polyclonal antibody (diluted 1:5000, ABclonal). Image J Program Analyzer (Media Cybernetics, USA) was used to quantify the protein band density.
6
Hippocampal Mitochondrial Dysfunction Protein Expression
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The hippocampal tissues were harvested at scheduled time points to test the expression levels of proteins indicating mitochondrial dysfunction and ER stress, including Bax, Bcl-2, caspase 3, GRP78, and CHOP. Equal amounts of protein from each sample were separated by using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto nitrocellulose membranes. The blots were blocked with 5% non-fat dry milk in TBS solution and probed with Bax (ab32503), Bcl-2 (ab185002), caspase 3 (ab197202), GRP78 (ab21685), CHOP (ab11419), and p-NF-κB p65 (ab222494) antibodies (diluted 1:1,000, Abcam) overnight at 4°C. The membranes were then washed and incubated with a fluorophore-conjugated donkey anti-rabbit secondary antibody (diluted 1:10,000, Rockland), which underwent excitation with light (700/800 nm). The emitted light was then detected and analyzed by using an Odyssey gel imaging system (LI-COR, Inc., Lincoln, NE, USA). β-Actin expression was analyzed in the same blots by using a monoclonal antibody (BS6007M, diluted 1:1500; Bioworld Technology). Relative expression was quantified with respect to the signals of the corresponding β-actin band.
7
Protein Extraction and Immunoblotting Protocol
8
Immunoblot Analysis of Optineurin
9
SDS-PAGE and LC-MS/MS Analysis of Proteins
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The total protein sample was separated on a 12% gel via SDS-PAGE and stained with a Coomassie Brilliant Blue staining solution, and then decolourized until the blue background was removed. The differential protein bands in the aconitine group were analysed using the Odyssey gel imaging system (LI-COR Biosciences, USA). After reduction and alkylation, trypsin was used to hydrolyse the differentially expressed proteins. Finally, LC-MS/MS was used to analyse the samples. The qualitative information of the target proteins was obtained using the MASCOT software.
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